Integrins are cell surface receptors that play a key role in morphogenetic events during development, and in a variety of pathogenic conditions, such as metastasis. The long-term goal of this project is to understand how integrin function is modulated during differentiation. Our work to date has focused predominantly on the B1 integrin subunit and the functional role of phosphorylation on a serine residue in its cytoplasmic domain. Specific aims will continue to study the functional significance of Bl phosphorylation and to examine how this phosphorylation event is regulated. In addition, we will begin to study the role of tetraspanin proteins in modulating integrin function. Specific Aim 1: To determine the role of serine phosphorylation of the cytoplasmic domain in B1 function. Preliminary studies suggest that cells expressing B1 with amino acid substitutions that mimic a phosphorylated serine in the cytoplasmic domain behave differently from cells expressing the wild type serine or neutral amino acids substitutions at this site. We will extend these studies using the 131-deficient parental GD25 fibroblast cell line and transfected cell lines expressing site-specific mutations. We will also investigate the ability of wild type and the site-specific mutant forms of BI to associate with a variety of focal adhesion proteins using a number of experimental approaches including co-immunoprecipitation and GST-pull down experiments. Specific Aim 2: To investigate how serine phosphorylation of the B1 integrin subunit is regulated in F9 cells. Our preliminary studies suggest roles for the phosphatase PP2A and the kinase ILK. Approaches will include determining if phosphorylated B1 can be de-phosphorylated in vitro by PP2A and investigating the behavior of F9 cells either overexpressing ILK or expressing a dominant negative form of the kinase. Specific Aim 3: To examine the role of the tetraspanins CD81 and CD9 in a6B1-mediated cell migration. These tetraspanins associate with a6B1, and CD9 appears to assist integrin-mediated sperm-egg fusion. We will first determine if these tetraspanins associate with the integrin in F9 cells using co-immunoprecipitation analysis. We will then use function-blocking antibodies to test the relative roles of these proteins in cell migration.